O comércio mundial de enzimas industriais estava estimado em 2.3 bilhões de dólares entre detergentes (U$ 789 milhões), aplicações alimentícias (U$ 634 milhões), agricultura (U$ 237 milhões), entre outros. Neste contexto, as enzimas lipolíticas estão atraindo enorme atenção devido ao seu potencial biotecnológico, visto que estas podem catalisar múltiplas reações (hidrólise, acidólise, interesterificação e glicerólise). Enzimas lipolíticas de origem microbiana são economicamente atrativas por serem biodegradáveis, atuarem normalmente em condições brandas, e serem quimio-seletivas propiciando à indústria farmacêutica a obtenção de drogas com efeito colateral reduzido. Neste projeto, quatro genes potenciais codificadores de esterases/lipases, advindos de uma biblioteca metagenômica de um consórcio microbiano degradador de óleo diesel, foram clonados em vetores de expressão e expressos em Escherichia coli BL21 (DE3), e as proteínas correspondentes foram submetidas a ensaios funcionais e estruturais. / The global trade of industrial enzymes is estimated at 2.3 billion U.S. dollars, divided mainly between detergents (US$ 789 million), food applications (US$ 634 million), and agriculture (US$ 237 million). Within this trade, lipolytic enzymes have attracted enormous attention because of their biotechnological potential as catalysts of multiple reaction types (including hydrolysis, acidolysis, interesterification and glycerolysis). Lipolytic enzymes of microbial origin are economically attractive because they are easily biodegradable, usually act in mild conditions, and are chemo-selective, providing the pharmaceutical industry a method for obtaining drugs with reduced side effects. In this project, four individual genes encoding putative esterases/lipases identified in a metagenomic library obtained from a microbe consortium isolated from diesel oil-contaminated soil were cloned into expression vectors and expressed in Escherichia coli BL21 (DE3), and their corresponding recombinant proteins were used for functional and structural studies.

No description available.

Thesis (Ed. D.)--Indiana University of Pennsylvania. / Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-02Bitstream added on 2014-06-13T19:55:59Z : No. of bitstreams: 1 paixao_daa_me_jabo.pdf: 1354562 bytes, checksum: 375f9e3806bd3d094111e5d1f95f8ddd (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A estratégia de clonagem e sequenciamento do gene 16S rRNA é uma das técnicas moleculares que permite estimar e comparar a diversidade microbiana de diferentes amostras ambientais. O objetivo deste trabalho foi estimar a diversidade de microrganismos pertencentes ao Domínio Bactéria em um consórcio degradador de óleo diesel, por meio do sequenciamento parcial do gene 16S rRNA, assim como desenvolver uma nova metodologia de rastreamento em bibliotecas metagenômicas. O consórcio bacteriano foi obtido através de solo enriquecido com óleo diesel. O DNA metagenômico foi extraído com o auxílio do kit Fast DNA spin Kit for soil (Bio101- Quantum Biotechnologies) e amplificado por uma reação de PCR (Reação em Cadeia da Polimerase) com os oligonucleotídeos iniciadores FD1 e RD1 específicos para a o gene 16S rRNA. Os produtos de PCR foram clonados em vetor pGEM T Easy (Promega) e transformados em células competentes de Escherichia Coli DH5 . O sequenciamento parcial dos clones foi feito com oligonucleotideos universais do vetor. Para a prospecção gênica foi utilizado membranas de nylon com “pools” de DNA de todas as placas. A biblioteca obtida gerou 431 clones. Os clones obtidos apresentaram similaridade com o filo Proteobacteria, com representantes das classes Gammaproteobacteria, Alphaproteobacteira e Betaproteobacteria. O gênero Pseudomonas apresentou-se com maior frequência de clones na biblioteca. O “software” DOTUR foi usado para determinar o número de unidades taxonômicas operacionais (OTUs). A curva de extinção indicou que os 431 clones sequenciado foram suficientes para estimar a diversidade bacteriana do consórcio. A metodologia testada baseado em “pools” de DNA foi eficiente na detecção e isolamento do gene Alkb na bilbioteca metagenomica. / Cloning and sequencing of 16S rRNA gene it is one of the molecular techniques that permits estimate and compare the microbial diversity of different environmental samples. The aim of this work was estimate the diversity of microorganisms that belong to Bacteria domain in a consortium specialized in diesel oil degradation, through partial sequencing of 16S rRNA gene, as well as develop a new methodology for screening libraries in metagenomics. This consortium was obtained through enrichments achieved using diesel oil in soil samples. The metagenomic DNA was obtained using Fast DNA spin Kit for soil (Bio 101-Quantum Biotechnologies) and amplified by PCR (Polymerase chain reaction) with FD1 and RD1 oligonucleotides, which are specific for 16S rRNA gene. The PCR products were cloned into pGEM-TEasy vector (Promega) and Escherichia coli DH5 was used as the host cell for recombinant DNAs. The partial clones sequencing was obtained using universal primers of the vector. For the exploration of gene were used nylon membranes with Pools of DNA from all plates. The library generated from 431 clones. All clones sequenced showed similarity with the phylum Proteobacteria, distributed in Gammaproteobacteria, Alphaproteobacteira and Betaproteobacteria classes. The Pseudomonas genus was the most abundant genus found in the metagenomic library. The DOTUR software was used to assigns sequences to operational taxonomic units (OTUs). Using the OTUs composition data, rarefaction curves were made to show that 431 sequences were enough to obtain a satisfactory coverage of diversity of the microbial consortium. The test methodology based on Pools of DNA isolation was effective in detecting the gene Alkb Library metagenomics.

Orientador: Eliana Gertrudes Macedo Lemos / Banca: Alessandro Minillo / Banca: Janete Apparecida Desidério Sena / Resumo: A estratégia de clonagem e sequenciamento do gene 16S rRNA é uma das técnicas moleculares que permite estimar e comparar a diversidade microbiana de diferentes amostras ambientais. O objetivo deste trabalho foi estimar a diversidade de microrganismos pertencentes ao Domínio Bactéria em um consórcio degradador de óleo diesel, por meio do sequenciamento parcial do gene 16S rRNA, assim como desenvolver uma nova metodologia de rastreamento em bibliotecas metagenômicas. O consórcio bacteriano foi obtido através de solo enriquecido com óleo diesel. O DNA metagenômico foi extraído com o auxílio do kit Fast DNA spin Kit for soil (Bio101- Quantum Biotechnologies) e amplificado por uma reação de PCR (Reação em Cadeia da Polimerase) com os oligonucleotídeos iniciadores FD1 e RD1 específicos para a o gene 16S rRNA. Os produtos de PCR foram clonados em vetor pGEM T Easy (Promega) e transformados em células competentes de Escherichia Coli DH5 . O sequenciamento parcial dos clones foi feito com oligonucleotideos universais do vetor. Para a prospecção gênica foi utilizado membranas de nylon com "pools" de DNA de todas as placas. A biblioteca obtida gerou 431 clones. Os clones obtidos apresentaram similaridade com o filo Proteobacteria, com representantes das classes Gammaproteobacteria, Alphaproteobacteira e Betaproteobacteria. O gênero Pseudomonas apresentou-se com maior frequência de clones na biblioteca. O "software" DOTUR foi usado para determinar o número de unidades taxonômicas operacionais (OTUs). A curva de extinção indicou que os 431 clones sequenciado foram suficientes para estimar a diversidade bacteriana do consórcio. A metodologia testada baseado em "pools" de DNA foi eficiente na detecção e isolamento do gene Alkb na bilbioteca metagenomica. / Abstract: Cloning and sequencing of 16S rRNA gene it is one of the molecular techniques that permits estimate and compare the microbial diversity of different environmental samples. The aim of this work was estimate the diversity of microorganisms that belong to Bacteria domain in a consortium specialized in diesel oil degradation, through partial sequencing of 16S rRNA gene, as well as develop a new methodology for screening libraries in metagenomics. This consortium was obtained through enrichments achieved using diesel oil in soil samples. The metagenomic DNA was obtained using Fast DNA spin Kit for soil (Bio 101-Quantum Biotechnologies) and amplified by PCR (Polymerase chain reaction) with FD1 and RD1 oligonucleotides, which are specific for 16S rRNA gene. The PCR products were cloned into pGEM-TEasy vector (Promega) and Escherichia coli DH5 was used as the host cell for recombinant DNAs. The partial clones sequencing was obtained using universal primers of the vector. For the exploration of gene were used nylon membranes with Pools of DNA from all plates. The library generated from 431 clones. All clones sequenced showed similarity with the phylum Proteobacteria, distributed in Gammaproteobacteria, Alphaproteobacteira and Betaproteobacteria classes. The Pseudomonas genus was the most abundant genus found in the metagenomic library. The DOTUR software was used to assigns sequences to operational taxonomic units (OTUs). Using the OTUs composition data, rarefaction curves were made to show that 431 sequences were enough to obtain a satisfactory coverage of diversity of the microbial consortium. The test methodology based on Pools of DNA isolation was effective in detecting the gene Alkb Library metagenomics. / Mestre

Made available in DSpace on 2017-07-10T17:47:58Z (GMT). No. of bitstreams: 1 Carolina_Aleteia_Mecabo.pdf: 1439256 bytes, checksum: c24dcfc3f28c7c0959b8b9303316bd0d (MD5) Previous issue date: 2014-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The study was conducted at Embrapa Beef Cattle, Campo Grande, Mato Grosso do Sul State, with the objective of evaluating the growth and nutritive value of grass in Piata agrosilvopastoral system with two densities of eucalyptus: 22 and14 meters between rows of eucalyptus. The experimental design was a randomized block design in a split plot design with four replications during winter. The plots treatments were the Integration Cattle Farming Forest at systems 1 and 2, which consisted of Brachiaria brizantha piatã planted after soybean harvest in the cropping system in consortium with eucalyptus (Eucalyptus urograndis) with 2m spacing between trees and between rows of 22 and 14 m, and the subplot treatments, points A, B, C, D and E, which were arranged between the rows of trees. The forage growth was evaluated, performing sampling 50 days after emergence, and every 15 days at each point (A, B, C, D and E) within each plot. During this period, also proceeded to analyze the morphogenesis of grass for 60 days with weekly reviews 5 tillers were marked within each section (A, B, C, D and E) within each portion. The variables analyzed were canopy height, canopy cover, plant density, leaf area index, leaf/stem ratio, dry matter production, Spad index, percentage of shade and morphogenesis (final leaf length, leaf appearance rate, rate of leaf elongation, stem elongation rate, lifespan of leaves and phyllochron number of live leaves per tiller). After the establishment of forage, which occurred at 110 days, the first court was held, where the entire sample was collected from each sub plot for the evaluation of the total production of forage and morphological components of nutritional assessment. There was a significant density of Eucalyptus for phyllochron variable. For the lifespan of leaves and plant density did not affect variable and this is not a variable influenced by shading. The dry matter content of the forage was the only variable that was higher in the near trees, where the highest percentage of shading points were found. The other morphogenetic and structural variables evaluated had a strong negative influence of shading, with lower values at the closest points to the trees. Treatment with 22 meters of distance between rows of trees, with a density of 227 eucalyptus trees/ha showed the best results of nutritional composition / O estudo foi desenvolvido na Embrapa Gado de Corte, em Campo Grande, no Estado do Mato Grosso do Sul, com o objetivo de avaliar o crescimento e o valor nutritivo do capim-piatã em sistema agrossilvipastoril com duas densidades de eucalipto: 22 e 14 metros entre fileiras de eucalipto. O delineamento experimental foi o de blocos casualizados em esquema de parcelas subdivididas, com quatro repetições, durante o inverno. Os tratamentos das parcelas foram os sistemas Integração Lavoura Pecuária Floresta 1 e 2, que consistiram no capim Brachiaria brizantha cv. Piatã, plantado após a colheita da soja, no sistema de cultivo em consórcio com o eucalipto (Eucalyptus urograndis), com espaçamento entre árvores de 2 m, e entre fileiras de 22 e 14 m, e os tratamentos das subparcelas, os pontos A, B, C, D e E, que eram marcados entre as fileiras das árvores. Avaliou-se o crescimento da forrageira, realizando-se amostragens 50 dias após a emergência, e a cada 15 dias, em cada ponto (A, B, C, D e E) dentro de cada parcela. Durante esse período, procedeu-se também a análise da morfogênese do capim, durante 60 dias, com avaliações semanais. Foram marcados 5 perfilhos dentro de cada ponto (A, B, C, D e E), dentro de cada parcela. As variáveis analisadas foram altura do dossel, cobertura do dossel, densidade de plantas, índice de área foliar, relação folha/colmo, produção de matéria seca, índice Spad, porcentagem de sombra e morfogênese (comprimento final de folha, taxa de aparecimento de folhas, taxa de alongamento de folhas, taxa de alongamento de colmo, duração de vida das folhas, filocrono e número de folhas vivas por perfilho). Após o estabelecimento da forragem, que se deu em 110 dias, foi realizado o primeiro corte, onde foi coletada toda a amostra de cada subparcela, para a avaliação da produção total, de componentes morfológicos da forragem e avaliação nutricional. Houve efeito significativo da densidade de eucalipto para a variável filocrono. Para a variável duração de vida das folhas e densidade de plantas não houve efeito significativo, não sendo esta uma variável influenciada pelo sombreamento. O teor de matéria seca da forragem foi a única variável que foi maior nos pontos mais próximos às arvores, ou seja, onde foram encontradas as maiores porcentagens de sombreamento. As demais variáveis morfogênicas e estruturais avaliadas tiveram forte influência negativa do sombreamento, sendo seus valores menores nos pontos mais próximos às árvores. No tratamento com 22 metros de distância entre fileiras de árvores, com densidade de eucalipto com 227 árvores/ha apresentou os melhores resultados de composição nutricional

Philosophiae Doctor - PhD / South Africa’s transition from apartheid to democracy necessitated transformation within all sectors to ensure their appropriateness for the new democratic era. In line with the national transformation agenda and the transformation and restructuring of the higher education sector, the Minister of Education in 2002 announced that the University of the Western Cape (UWC) and the Cape Peninsula University of Technology (CPUT) would be the only enrolling institutions for undergraduate nursing education in the Western Cape. This decision meant that the University of Stellenbosch and the University Cape Town would no longer enrol undergraduate nurses, but would combine their strengths in a collaborative manner with UWC to train nurses for the region. The Cape Higher Education Consortium (CHEC), however, proposed the establishment of a Common Teaching Platform (CTP) for undergraduate nursing education in the region, requiring collaboration between all higher education institutions in the Western Cape. The Common Teaching Platform came into effect in 2005. The purpose of the study was to evaluate the effectiveness of the regional collaboration on the Common Teaching Platform for B Cur Nursing in the Western Cape. An evaluation research design using qualitative methods was adopted for the study. Stufflebeam’s decision-oriented evaluation model, which caters for the evaluation of the context, input, process and product components of programmes, was used to guide the research process. Semi-structured, in-depth interviews, focus group discussions and a record review were used to collect data from the Chief Executive Officers of CHEC; Deputy Vice-Chancellors of the participating universities; Deans of the Health Science Faculties; Heads of Departments, Lecturers and Students of the Nursing Departments of the participating universities. The study adopted an inductive approach to data analysis. The inductive analysis procedure described by Thomas (2003) was adapted and used. The results evinced a general lack of application of the basic tenets of change management and a systems approach to the planning and implementation of the Common Teaching Platform. Transformation of nursing education in the Western Cape, according to the results, was in line with the national transformation agenda. Participants, however, felt that people were not yet ready to collaborate and needed enough time to accept the change, given that transformation was relatively new in the country. A critical finding was that important stakeholders were excluded from the planning phase, which led to challenges during the implementation of the Common Teaching Platform. The results further highlighted that a top-down approach was adopted. Numerous challenges with regards to the implementation of the Common Teaching Platform, including inter alia, poor communication, lack of commitment to the collaboration process, lack of adequate resources and challenges with the delivery of the curriculum, were shared by all the participants. Despite all these challenges the results showed that the student throughput rates were not compromised, and that the number of reported complaints from lecturers and students decreased over the years. On the whole, however, participants felt that the goals of the collaboration were not met due to the unresolved challenges which included inadequate resources, lack of sharing of resources and expertise across institutions, lack of commitment to participation on the CTP and failure to produce sufficient graduates to address the nurse shortage in the province. / National Research Foundation (NRF) and The Atlantic Philanthropies

The utilisation of fossil fuels for energy worldwide depletes the natural reserves and at the same time releases billions of tonnes of carbon dioxide and other greenhouse gases into the atmosphere. In order to reverse the negative effects of this accumulation, i.e., global warming and climatic changes, countries around the world are now considering nuclear energy and other cleaner sources of energy as a substitute to the burning of fossil fuels. The deployment of the later technology has progressed slowly due to lack of public support. The general public and environmental lobbyists worry about the discharge of radioactive waste from nuclear power generation and accidents that have occurred in the nuclear power industry in the recent past. One of pollutants of concern is uranium which is discharged from the nuclear generation processes as the highly toxic uranium-6, (U(VI)). U(VI) coming from the reactors is radioactive as well as highly toxic to aquatic life forms. Biological treatment of metal pollutants is viewed as an environmentally friendly alternative to conventional physical/chemical treatment methods, especially in dilute solutions where physical/chemical methods may not be effective. Microbial processes may be applied both as in situ and/or ex situ processes. Microbial consortia, consisting of several species of microorganisms in the form of bioflocs for reducing/removing the pollutants have been used as they preserve the complex interrelationships that exist between species in the source. The results of this study demonstrate the potential of microbial U(VI) reduction as a possible replacement technology for physical/chemical processes currently in use in the nuclear industry. A detailed analysis of the biological reduction of uranium-(VI) was conducted and the following were the main findings of the study: (1) Background uranium concentration in soil from the mine was determined to be 168 mg/kg, a very high value compared to the typical concentration of uranium in natural soils; (2) Among six bacteria species isolated from a uranium mine in Limpopo, South Africa, three anaerobic species – Pantoea sp., Enterobacter sp. and Pseudomonas stutzeri – reduced U(VI) to U(VI) and facilitated the removal of the uranium species from solution. Based on batch studies and cell disruption studies, the laws governing microbial U(VI) reduction were determined and the kinetic parameters for U(VI) reduction were determined. The cultures in this study reduced uranium-U(VI) at a rate better than rates found in literature for other microorganisms. Reduction rates reported in this paper can be used to assess the applicability of bioreduction for uranium removal processes. / Dissertation (MSc)--University of Pretoria, 2011. / Chemical Engineering / unrestricted

The results of this study provide support for the use of the SERVQUAL and market orientation instruments for evaluating agencies approach to service delivery issues in Taiwan. The results indicate that on two of the five dimensions of the SERVQUAL instrument in particular have the potential for identifying areas of concerns and facilitating focusing on specific concerns and developing mechanisms for addressing these concerns. These areas are responsiveness and reliability. The potential for improving the applicability and sophistication of the instrument also exists. There are clearly issues that can be addressed between the perceptions and expectations of both customers and managers for the advertising agencies. In particular, the results indicate that customers have a much bigger gap between expectations of service and the delivery of service than the managers. This research provides evidence that a service problem exists in advertising agency settings in Taiwan as well as a mechanism for identifying and measuring the extent of the problem. In addition, the consortium based advertising agencies had a different market orientation than the matrix or integrated organizations studied. Consortium based organizations are more likely to have a competitor orientation while matrix and integrated organizations are more likely to have a customer orientation. In addition, market orientation impacts attitudes and approaches to service quality.

O comércio mundial de enzimas industriais estava estimado em 2.3 bilhões de dólares entre detergentes (U$ 789 milhões), aplicações alimentícias (U$ 634 milhões), agricultura (U$ 237 milhões), entre outros. Neste contexto, as enzimas lipolíticas estão atraindo enorme atenção devido ao seu potencial biotecnológico, visto que estas podem catalisar múltiplas reações (hidrólise, acidólise, interesterificação e glicerólise). Enzimas lipolíticas de origem microbiana são economicamente atrativas por serem biodegradáveis, atuarem normalmente em condições brandas, e serem quimio-seletivas propiciando à indústria farmacêutica a obtenção de drogas com efeito colateral reduzido. Neste projeto, quatro genes potenciais codificadores de esterases/lipases, advindos de uma biblioteca metagenômica de um consórcio microbiano degradador de óleo diesel, foram clonados em vetores de expressão e expressos em Escherichia coli BL21 (DE3), e as proteínas correspondentes foram submetidas a ensaios funcionais e estruturais. / The global trade of industrial enzymes is estimated at 2.3 billion U.S. dollars, divided mainly between detergents (US$ 789 million), food applications (US$ 634 million), and agriculture (US$ 237 million). Within this trade, lipolytic enzymes have attracted enormous attention because of their biotechnological potential as catalysts of multiple reaction types (including hydrolysis, acidolysis, interesterification and glycerolysis). Lipolytic enzymes of microbial origin are economically attractive because they are easily biodegradable, usually act in mild conditions, and are chemo-selective, providing the pharmaceutical industry a method for obtaining drugs with reduced side effects. In this project, four individual genes encoding putative esterases/lipases identified in a metagenomic library obtained from a microbe consortium isolated from diesel oil-contaminated soil were cloned into expression vectors and expressed in Escherichia coli BL21 (DE3), and their corresponding recombinant proteins were used for functional and structural studies.